his-tag purification - (Jan/09/2014 )
hello,
may anyone guide me in this purification porblem as attached. Do i doing any mistake?
Well, you're using an imidazole that strongly absorbs at A280 and so you won't be able to see an elution peak even if it were there. I had the same problem the first time I did a his purification on an AKTA. Even though the imidazole I purchased was of high purity I needed to go back to Sigma and purchase one that spoke to A280 absorbance in its characteristics. More pricey but I think it's worth it. Alternatively, it's not too much more expensive (less than $20 if my memory is correct) to buy the His Buffer Kit from GE that contains 200ml of 8x sodium phosphate/NaCl buffer and 100ml of 2M imidazole that does not absorb.
My bigger concern is that your flow thru peak is tiny. Are you sure that there is protein in your initial sample? What size injection loop are you using? More details about the injection and loading of your column and sample prep and I may be able to help....
Your problem is that there is no protein in your sample, or your sample with protein never made it through the FPLC. My guess is that when you injected your sample it went right to the waste.
I would also suggest keeping your NaCl concentration the same in both binding and elution buffers. You will need to dialyze, desalt or gel filter to get rid of the imidazole anyways, so exchange for lower NaCl at that time. This isn't the source of your problem though, but is only a suggestion.
I'm assuming you used a sample loop but the details around the loop and how you are injecting could be responsible for your low level of protein....
i'm using 2 ml sample loop..
based on my screening on sds-page n western blot, targetted band have been acqiured even a poor band observed.
actually, i need to run IMAC, but AKTAprime here is so old, purchased since 2001 and i could not find IMAC protocol in this mechine.
thus, i run ion exchange gradient elution protocol.
the column i used is HisTrap HP 5ml..
i run system wash and wash flow path for distiiled water and binding buffer.
for wash flow path i inject distilled water and binding buffer at the end of cycle
here my setting
Manual run>set Method Base (ml)> set concentration B(0%)>set gradient (off)>set flow rate (1ml/min)>set fraction base (ml)>set fraction size (0 ml)> set pressure limit (0.37 MPa)>set buffer valve position (Pos 1)> set Inject Valve Position(load). start Run
for end point setting, it like as file attached.
I also use an old AKTA and run the ion exchange gradient elution only with a HisTrap FF 1ml column.
I'm confused by your settings. Are you not loading the column (injecting) as part of the ion exchange gradient elution protocol? If not, that could be your problem.
thank you missle, my reading on pressure limit with and without column installed give the same reading value?
is it leading to this purification problem?
Dear Ahmadfathi,
Based on your attached chromatogram, i could be wrong, but still, my suspect is that you did not perform a proper system wash, based on your conductivity curve.
Your conductivity curve should comes low and gradually increasing during your elution, as your buffer imidazole concentration gradually increase.
This could be due to the possibility that you are running the flow path wash with buffer B (or some other buffer else) instead of buffer A (binding buffer).
I would always suggest you run your binding buffer till the conductivity curve stabilized.
Also, can you explain your configuration of "buffers position 2"? Do you put two different buffer in the valve-A?
Your sample buffer is better not to use with Tris-EDTA-Sucrose. You should suspend your buffer in Binding buffer by dialysis or using desalting column to do buffer exchange. Better still, totally omit imidazole in your binding buffer for your first experiment. You only put in minor imidazole when you need a more pure elution later.
To fully fill in your sample loop, you need to put at least 2x of the sample loop volume, due to flow dynamics. For your case, you need to fill in at least 4ml of the sample volume before you run your AKTA. If you afraid of sample lost, you can use partial loading method.
For your system pressure, it should be maintained under 0.3Mpa, that is to make sure your column doesn't break and also maintain your resin's structure. I think 0.37Mpa of your setting should be fine. Since your column 5ml column suppose to be able to run up to 5ml/min, running 1ml/min will less likely to increase the system pressure, which explains the reason of your system pressure will less likely to build up with or without column.
As suggested by missle, you can use IEX profile to run your IMAC protocol.
Hope this helps, and good luck.
Rgs,
Adrian
p/s: you can send over your AKTAprime configuration txt file.
Adrian K on Wed Jan 15 06:57:46 2014 said:
To fully fill in your sample loop, you need to put at least 2x of the sample loop volume, due to flow dynamics. For your case, you need to fill in at least 4ml of the sample volume before you run your AKTA. If you afraid of sample lost, you can use partial loading method.
you can also half fill your sample loop (1ml) so that you don't waste any sample. you'll just have to make more runs or, if the column has the capacity, more load steps per run.
I think that your biggest problem is that your sample is not going over the column (or there is barely protein in the sample). Because the UV reading is being taken after the buffer has flowed thru the column I would say the pressure observation isn't the problem.
In regards to the volume to load, the following is a link to GE's training document (search for 'dynamics' within the document and you'll find it) that explains nicely the sample loop issue. Personally, I wouldn't put 2x the loop volume as you'll be sending 30%+ of your sample straight to waste. I typically only fill the loop to 75% capacity so I don't waste my sample and then program the sample injection volume (in the ion exchange gradient elution protocol) to be 150% the capacity of the sample loop so that the loop is flushed with binding buffer and that the excess 'flushed sample' goes over my column.
I agree with Adrian K that you should be wary of the EDTA in your sample buffer. Dialyzing is the most complete way of handling the problem but I've also had success diluting my sample with concentrated binding buffer so that the EDTA is within column tolerable limits and so my sample has the appropriate amount of NaCl (typically 0.5M) and imidazole (~20mM) as the column manufacturer recommends.
Below is an example of a typical purification for me. I hope this helps and isn't more info than you were looking for. It doesn't address all the potential issues but I know when performing the method this way all my sample goes over the column...
"Sample Preparation – To the ~3ml lysate, add 430ul 8x Sod. Phos. NaCl buffer, 35ul 2M Imidazole. The sample was centrifuged and not filtered prior to loading over AKTA. Care was taken to transfer supernatant without disturbing the pellet.
Column – HisTrap 1ml Column (lot# 10150414) – 1st use
Purification:
§ Purge line B with dH2O at 50ml/min to waste. Load over column at 1ml/min until baseline.
- Purge w/ Elution Buffer to waste.
- Purge line A1 with dH2O at 50ml/min to waste.
- Purge line A1 with binding buffer at 50ml/min to waste. Load over column at 1ml/min until baseline.
- Purge 5ml sample loop with dH2O followed by binding buffer using a 20cc syringe
- Inject ~3.5ml prepared sample into the sample loop via the injection port.
§ Program method (Ion Exchange/Gradient Elution) - see below.
- Collect flow thru peak
§ When method is complete: rinse column w/ 3-5 CV of dH2O.
- Equilibrate column in 20% ethanol.
Method:
Select Template/Method Template/ Ion Exchange/Gradient Elution
Sample Inject: Inject
Pressure Limit: 0.6 mPa
Flow Rate: 0.3 ml/min *Increase flow rate to 1ml/min after sample is fully loaded to speed process
Fraction Size: 1.0 ml
Equilibr. Vol.: 0.5 ml
Sample Inj. Vol.: 6 ml (3.5ml sample)
Wash 1 Vol.: 20ml
Elution Vol.: 20ml
Wash 2 Vol.: 3 ml (method adds ~12ml) "